ABSTRACT DNA damage-mediated genome instability is a contributing factor in the causation of human diseases, including neurodegeneration, immunological disorders, and cancer. Understanding how cells prevent and manage DNA damage is highly significant. R loop, a transcription-linked 3 nucleic-acid structure consisting of a RNA:DNA hybrid and a displaced single-strand DNA (ssDNA), when decorated with histone H3S10p mark causes genomic instability upon its cleavage into DNA double strand breaks (DSBs). Currently, nothing is known about R loop dysfunction in the causation of human primary immunodeficiency disorders (PIDDs), aka, inborn errors of immunity. Using a PID disease model of human Wiskott-Aldrich syndrome (WAS), we recently discovered an essential nuclear role of WASp, the protein deficient in WAS, in limiting R loop-mediated DNA damage in CD4+ T helper 1 (Th1) lymphocytes. This discovery has opened up a new avenue of research into how WASp, a nucleocytoplasmic protein with dual-roles in F-actin polymerization and gene transcription, ensures R loop-linked genome stability. The current proposal seeks to define the nuclear signaling pathways and mechanisms involved in ensuring a healthy R loop balance, and therefore a stable genome, in human Th cells, and how their disruptions by WAS gene mutations is causally-linked to the development of immune deficiency and clinical phenotypes in WAS. Aim 1 focuses on defining chromatin-based mechanisms by which WASp influences the balance between beneficial (?good?) R loops and deleterious (?bad?) R loops, and their effects in the causation of WAS Th1 and WAS Th2 cellular phenotypes. Aim 2 will clarify the mechanism of the newly identified mRNA splicing defect in WAS Th cells as it relates to R loop formation and genome instability. Aim 3 will utilize primary T cells from multiple WAS patients of differing clinical severities to establish R loop load in the T cells as a ?dynamic? disease biomarker, and to define the involvement of nuclear-F-actin effects of WASp in WAS phenotype development. In the long-term, the knowledge gained from these studies will foster the development of novel prognostics, diagnostics, and therapeutics for this PID and other R loop-mediated immunological disorders.